The quantitative analysis of ammonia producing substances such as creatinine and urea etc. which are contained in biological fluids is of the utmost importance in the diagnosis of diseases such as kidney disease, in examinations carried out for controlling the course of such diseases and in the examination of kidney function.
Method of analysis by a procedure in which ammonia is produced from an ammonia producing substance and a procedure in which the ammonia so produced is determined are typical of the methods used for the analysis of ammonia producing substances. Such methods of analysis involving conversion to ammonia used on a wide scale in the past include wet methods or solution methods. Moreover, in recent years, dry methods of analysis based on conversion to ammonia in which dry analytical devices as typified by the integral multi layer analytical element are used, have been proposed and put into practice.
The above mentioned procedures for producing ammonia from ammonia producing substances generally involve methods in which the ammonia is generated by the action of an enzyme. For example, methods in which the creatinine in a liquid sample is hydrolyzed specifically to ammonia and N-methylhydantoin by means of creatinine deiminase (EC3.5.4.21) can be used for the quantitative analysis of creatinine in a biological fluid. Furthermore, methods in which urea is hydrolyzed to ammonia and carbon dioxide using urease as a catalyst can be used for the quantitative analysis of urea nitrogen in biological fluids (referred to below as BUN). In these methods the ammonia producing substance, which is the analyte, is the substrate of an enzyme and so they are known as ammonia generating substrates. Methods for the analysis of these ammonia generating substances have been disclosed in Analytical chemistry, 46, 246 (1974), Clinica Chimica Acta, 18, 409 (1967), Clinical Chemical Analysis III, Nitrogen Containing Components (Japanese) pages 13-14 and pages 67-87 (Tokyo Kagaku Dojin, Tokyo, 1979) and Journal of Medical Technology (Japanese, Vol. 5 (No. 6), pages 387-391 (1961) etc.
However, the biological fluids which are these analytes often contain ammonia (endogenous ammonia) in a state in which it has been released as ammonium ions, and it has not been possible to disregards the errors which have arisen as a result of the presence of this endogenous ammonia. Hence it is necessary to eliminate the effect of the endogenous ammonia in order to determine the quantity of the ammonia producing substrates precisely, and some methods for the elimination of endogenous ammonia have already been proposed.
Actual examples of methods for the analysis of ammonia producing substances which include a procedure for the pre-elimination of endogenous ammonia in a wet method include the quantitative methods disclosed in Japanese Patent Application (OPI) Nos. 5,198/83 corresponding to GB 2102 568A, 21,398/84, 31,696/84, 31,697/84, 31,698/84, 31,700/84 and 56,095/86 corresponding to EP 0173 276A etc. (the term "OPI" as used herein refers to a "published unexamined Japanese patent application").
Integral multi-layer analysis elements for the analysis of ammonia producing substances in which the effect of the endogenous ammonia is eliminated (or abated) by an endogenous ammonia trapping function within the analysis element itself, such as the integral multi-layer elements disclosed in Japanese Patent Application (OPI) No. 278,761/86 corresponding to EP 0204 334A, have also been disclosed as dry methods of analysis. These multi-layer analysis elements have an endogenous ammonia trapping layer, in which an ammonia trapping reaction takes place, established adjacent to and above the layer in which ammonia is formed by the ammonia producing reaction.
More, reagent compositions which contain an enzyme which converts ammonia as substrate to another substance are included in the endogenous ammonia trapping layer. Actual examples of such compositions include those which contain NADH (the reduced form of nicotinamide adenine dinucleotide) and/or NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate), glutamic acid dehydrogenase (EC1.4.1.3) and .alpha.-ketoglutaric acid (or a salt thereof).
It is evident that because the endogenous ammonia trapping layer is adjacent to the reaction layer in which the ammonia producing reaction takes place, in integral multi-layer analysis elements for the analysis of ammonia producing substances which have an endogenous ammonia trapping layer, the measurement sensitivity has tended to be lower than that of an element in which no endogenous ammonia trapping layer is used, since ammonia which has been produced by the ammonia generating reaction diffuses upwards and takes part in a side reaction in the endogenous ammonia trapping layer.